The experiments proposed in this application aim to contribute to the understanding of gene regulation and chromosome structure. In particular, they focus on a curious homology-based process, called transvection, in which the pairing of homologous chromosomal regions leads to changes in gene expression. The proposed studies use Drosophila, where homologous chromosomes are paired in somatic tissues and several cases of transvection have been confirmed. However, their reach goes far beyond Drosophila, since pairing of homologous chromosomal regions contributes to a variety of potent and essential regulatory mechanisms in diverse species, such as X-inactivation in mammals and meiotic silencing of unpaired DNA/chromatin in mammals and fungi. Furthermore, as transvection concerns the manner in which enhancers interact with promoters, the findings emerging from the studies described here will pertain to gene regulation in any organism. Relevance to human health: Human development is exquisitely sensitive to the presence of homology, as evidenced by homology-driven processes such as X- inactivation, parental imprinting, allelic exclusion, and other forms of monoallelism. These processes are essential for normal human development;when they go awry, severe abnormalities and disease ensue. As such, the studies proposed here should advance knowledge of the etiology of some forms of diseases and, therefore, the design of strategies to improve health. Specific Aims: Aim 1 focuses on the form of transvection that involves enhancer action in trans, probing the underlying mechanism for trans action and asking whether an enhancer can flip-flop between a cis-linked promoter and promoter in trans. Aim 2 addresses the physical nature of paired genes, asking how pairing can alter gene topology and chromatin structure. Finally, Aim 3 proposes the use of cell lines for chromatin analyses and a whole genome screen to identify genes involved in pairing. These aims use genetic and molecular biological tools, including whole organismal genetics, mutational analyses, gene replacement and transgene technologies, RNAi, cell culture, and strategies for probing chromatin structure. PUBLIC HEALTH RELEVANCE: Transvection is a curious process through which the pairing of homologous chromosomal regions leads to changes in gene expression. Our goal is to advance the understanding of transvection as well as homolog pairing, because a combined analysis of these processes will provide unique insights into how chromatin and chromosome structure regulate gene expression. Given the importance of gene regulation for human development and health, our studies will be broadly relevant in the medical arena.